Modifications to the differential display technique reduce background and increase sensitivity.
Modifications to the differential display technique reduce background and increase sensitivity.
复制标题
对差分显示技术的修改减少了背景并提高了灵敏度。
DOI：
10.1006/abio.1995.1243
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Bos,TJ
中科院分区：
文献类型：
--
作者：
Hadman,M;Adam,BL;WrightJr,GL;Bos,TJ
the phenol and the interface while leaving nucleic acids in the aqueous phase. After centrifugation, the upper, aqueous phase is discarded, along with most salts and nucleic acids. In the second step, the phenol phase is mixed with 2 vol of ether. Centrifugation results in a large upper phase containing the phenol-ether mix and a lower phase of about 50 pul containing the protein in the water that saturated the phenol. In the third step, a second extraction with ether is performed to eliminate residual phenol. Omission of this step often results in widening of the protein bands during SDS-PAGE. In the fourth step, the sample is dried in a centrifugal evaporator. It may then be dissolved in vvater or electrophoresis buffer as required.To evaluate the technique, protein from a mammalian cell extract was precipitated with either TCA or phenol–ether or by the chloroform-methanol procedure of Wes-sel and Flügge (2). The samples were then analyzed by SDS-PAGE followed by silver staining and quantitation by densitometric scanning (Fig. 1). A nonprecipitated sample is shown in the first lane. Parallel samples diluted to 150 gul or to 1 ml with water were subjected to each procedure. The samples precipitated with 20% TCA without carrier showed less than 50% recovery (lanes TCA). Phenol-ether precipitation showed about 80% recovery (lanes PE) and the procedure of Wessel and Flügge showed 80 and 66% recovery (lanes WF), respectively. Phenol–ether appeared to precipitate all protein bands equally well. Samples containing O. 5% Triton X-100, 0.5% SDS, or 1 M NaCl and samples of purified myosin, ß-galactosidase, serum albumin, ovalbumin, and carbonic anhydrase were also recovered efficiently by phenol-ether precipitation (not shown). We next determined whether more dilute protein sam-ples can also be precipitated quantitatively with phenol–ether. An extract containing proteins metabolically la-
